Moreno-Pérez et al. Malaria Journal 2013, 12:165http://www.malariajournal.com/content/12/1/165
RESEARCH Open Access
Characterizing PvARP, a novel Plasmodium vivaxantigenDarwin A Moreno-Pérez1,2, Ambar Saldarriaga1 and Manuel A Patarroyo1,2*
Abstract
Background: Plasmodium vivax continues to be the most widely distributed malarial parasite species in tropicaland sub-tropical areas, causing high morbidity indices around the world. Better understanding of the proteins usedby the parasite during the invasion of red blood cells is required to obtain an effective vaccine against this disease.This study describes characterizing the P. vivax asparagine-rich protein (PvARP) and examines its antigenicity innatural infection.
Methods: The target gene in the study was selected according to a previous in silico analysis using profile hiddenMarkov models which identified P. vivax proteins that play a possible role in invasion. Transcription of the arp genein the P. vivax VCG-1 strain was here evaluated by RT-PCR. Specific human antibodies against PvARP were used toconfirm protein expression by Western blot as well as its subcellular localization by immunofluorescence.Recognition of recombinant PvARP by sera from P. vivax-infected individuals was evaluated by ELISA.
Results: VCG-1 strain PvARP is a 281-residue-long molecule, which is encoded by a single exon and has anN-terminal secretion signal, as well as a tandem repeat region. This protein is expressed in mature schizonts and islocated on the surface of merozoites, having an apparent accumulation towards their apical pole. Sera fromP. vivax-infected patients recognized the recombinant, thereby suggesting that this protein is targeted by theimmune response during infection.
Conclusions: This study showed the characterization of PvARP and its antigenicity. Further assays orientatedtowards evaluating this antigen’s functional importance during parasite invasion are being carried out.
BackgroundMalaria is a tropical disease that causes millions of deathsper year around the world. The World Health Organiza-tion’s (WHO) Malaria Report 2011 indicated that therewere 216 million cases and 655,000 deaths, mainly in chil-dren aged less than five years [1]. In spite of the incidenceof cases worldwide and mortality index having becomesubstantially reduced by 17% and 25% between 2000 and2010, respectively, the figures regarding cases of malariacontinue to be alarming. This is due to two main aspectsimpeding the total eradication of the disease: a gradual in-crease of parasite strains which are resistant to anti-malarial drugs [2] and populations of the mosquito vectorwhich are insecticide-resistant [3].
* Correspondence: [email protected]ón Instituto de Inmunología de Colombia (FIDIC), Carrera 50 No. 26-20, Bogotá, Colombia2Universidad del Rosario, Calle 63D No. 24-31, Bogotá, Colombia
Plasmodium vivax stands out as the most widespreadparasite species causing malaria in humans; it is foundthroughout tropical and subtropical areas of the worldand causes the disease’s highest morbidity indices on theAsian and American continents [4]. Even though it hasbeen thought that P. vivax was a benign species, recentstudies have shown that infection caused by this parasitecould cause severe clinical symptoms [5,6], similar tothose found in Plasmodium falciparum infection, therebymaking it a potential menace.Synthetic vaccines have been considered a good choice
among control strategies when combating infectious dis-eases. Regarding malarial blood stages, vaccine develop-ment has been focused on the recombinant expression ofparasite antigens (MSP-1 [7-9] and AMA-1 [10,11] havingbeen the most studied) or on using synthetic peptides
entral Ltd. This is an Open Access article distributed under the terms of the/creativecommons.org/licenses/by/2.0), which permits unrestricted use,, provided the original work is properly cited.
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[12,13]; however, no fully effective vaccine against any spe-cies has been reported to date.Recent work has established that the key to achieving an
effective vaccine lies in blocking the interaction of parasiteligands which facilitate adhesion to target cell receptors[14]; this means that molecules localized on parasite surfaceand apical organelles (rhoptries and micronemes) must beidentified. Unfortunately, data regarding the P. vivax pro-teins involved in invasion of reticulocytes that have beenfunctionally characterized to date lag behind that availablefor their P. falciparum counterparts [15]. The foregoing hasbeen due to the difficulty of standardizing an in vitro cul-ture given poor reticulocyte recovery from adult humantotal blood [16]. Such experimental limitation has led toseveral study alternatives having been suggested; probabilis-tic techniques have been most useful when predicting pos-sible vaccine candidates. A recent study involving hiddenMarkov models for analyzing the transcriptome of theP. vivax Sal-1 strain’s intra-erythrocyte life-cycle has led tothe identification of 45 proteins that play a potential role ininvasion; the role in cell adhesion for 13 of them (localizedin merozoite rhoptries or on their surface) had previouslybeen determined [17]. It was particularly interesting that anasparagine-rich protein (ARP) was found, this being con-served throughout the Plasmodium genus [17]. Only itsP. falciparum orthologue has been described to date, calledthe apical asparagine-rich protein (PfAARP) [18]. ThePfAARP-encoding gene has a prominent expression patterntowards the last intra-erythrocyte parasite developmentstage (48 hours post-invasion), which has been shown byreal-time PCR and Northern blot. Antigenicity assays haveshown that the N-terminal protein’s region (PfAARP-N)obtained as a recombinant is recognized by antibodies frompatients who have been naturally infected by P. falciparum.Rabbit antibodies directed against PfAARP-N have beenable to significantly inhibit parasite invasion of RBCin vitro. The foregoing, together with an RBC binding assayinvolving the expression of the complete protein on COScell surface, has highlighted this antigen’s functional role inparasite binding to and invasion of target cells [18].The present study was thus aimed at characterizing the
asparagine-rich protein orthologue for PfAARP in P. vivax.Molecular biology assays and immunochemistry techniqueswere used to demonstrate Pvarp gene transcription, proteinexpression and localization, as well as the ability to inducean antigenic response in patients who had suffered episodesof P. vivax malaria.
MethodsSelecting the gene and designing the primers andsynthetic peptidesPvARP was selected, bearing in mind the in silico studyby Restrepo-Montoya et al. [17] of P. vivax proteinsplaying a potential role in invasion. The PlasmoDB [19]
database was then scanned to obtain the Pvarp gene se-quence from the Salvador 1 (Sal-1) reference strain and toanalyze adjacent genes’ synteny in different Plasmodiumspecies. Specific primers were designed manually usingGene Runner software (version 3.05). B-cell lineal epitopeswere predicted with AntheProt software [20] using the de-duced amino-acid (aa) sequence. A tBlastn analysis of thepredicted B-cell epitopes was then carried out to selectpeptide sequences exclusive for the P. vivax ARP.
Animal handlingThe experimental animals used were handled in accord-ance with Colombian Law 84/1989 and resolution 504/1996. Aotus monkeys kept at FIDIC’s primate station(Leticia, Amazon) were handled following establishedguidelines for the care and use of laboratory animals(National Institute of Health, USA) under the constantsupervision of a primatologist. All experimental proce-dures involving Aotus monkeys had been previously ap-proved by the Fundación Instituto de Inmunología'sethics committee and were carried out in agreementwith the conditions stipulated by CorpoAmazonia (reso-lution 00066, 13 September, 2006). An Aotus monkeywas experimentally infected with the Vivax ColombiaGuaviare 1 (VCG-1) strain and monitored daily to assessinfection progress throughout the entire study (up today 18) using Acridine Orange staining. The monkey wastreated with paediatric doses of chloroquine (10 mg/kg onthe first day and 7.5 mg/kg/day until the fifth day) andprimaquine (0.25 mg/kg/day from the third to the fifthday) at the end of the study to guarantee parasite clear-ance from total blood. Once experiments were over,CorpoAmazonia officers supervised the primate’s returnto its natural habitat in excellent health.
Isolating the Plasmodium vivax parasiteVCG-1 strain parasites were maintained in vivo accordingto previously described methodology [21]. A P. vivax-infected blood sample (3 mL) was passed through a dis-continuous Percoll gradient (GE Healthcare, Uppsala,Sweden) according to an already established protocol [22]for obtaining schizont-stage enriched parasite. The samplewas then used as RNA, genomic DNA (gDNA) and totalprotein source.
Extracting RNA and cDNA synthesisTotal RNA was extracted from the schizont-enrichedsample using the Trizol method and treated with RQ1(RNA-qualified) RNase-free DNase (Promega, Wisconsin,USA) according to the manufacturer’s recommendations.Complementary DNA (cDNA) was synthesized using aSuperScript III enzyme (RT+) (Invitrogen, California,USA) in the following conditions: 65°C for 5 min, 50°C for1 hour and 70°C for 15 min. An additional reaction
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without the SuperScript III enzyme (RT-) was made foruse as control. Following 15 min’ incubation at 37°C withRNase (Promega, USA) the product was stored at −70°Cuntil its later use.
Cloning, sequencing and bioinformatics analysisThe cDNA RT + and RT- samples, as well as the gDNAobtained using a DNA Wizard Genomic purification kit(Promega), were used as template in 10 μL PCR reactionscontaining 0.5 U/μL Accuzyme DNA polymerase (Bioline),1x AccuBuffer, 2 mM MgCl2, 0.5 mM dNTP, 0.5 μMprimers and DNAse-free water for completing the reactionvolume. Specific primers were designed for amplifying a re-gion containing the entire Pvarp gene (direct 5′- CATTTGATCAGAGACGAC -3′ and reverse 5′- TTGGCACTTTTGTCACGA -3′), or the encoding sequence without thesignal peptide (direct 5′- atgTGCAACACAAATGGGAAAA -3′ and reverse 5′- CACGCCAAACAGCTTCA -3′);the protein expression start codon was included in the dir-ect primer’s 5′ end. A set of primers which had been previ-ously designed for amplifying the Pvron1-a region (direct5′- atgGCGAAGGAGCCCAAGTG-3′ and reverse 5′- ATCCCTAGCAATGCTTCG -3′) [23] was used as control forcDNA contamination with gDNA. The PCR for the Pvarpgene began with a denaturing step at 95°C for 5 min,followed by 35 cycles at 95°C for 30 sec, 52°C for 10 secand 72°C for 1 min. Pvron1-a PCR began with a denaturingstep at 95°C for 5 min, followed by 35 cycles at 95°C for30 sec, 56°C for 10 sec and 72°C for 1.5 min. A WizardPCR preps kit (Promega) was used for purifying Pvarp geneamplicons obtained from independent PCRs done with theRT + sample, once quality had been evaluated by 1% agar-ose gel. Pure products were then ligated to the pEXP5 CT/TOPO expression vector and transformed in TOP10Escherichia coli cells (Invitrogen). Various clones weregrown to purify the plasmid, using an UltraClean mini plas-mid prep purification kit (MO BIO laboratories, California,USA); insert integrity and its correct orientation were con-firmed by sequencing using an ABI PRISM 310 geneticanalyzer (PE Applied Biosystems, California, USA). VCG-1strain PvARP was characterized in silico using SignalP 3.0[24], FragAnchor [25], XSTREAM [26], tools and theInterpro database [27] to search for secretion signal orGPI-anchor sequences, tandem repeats and putative do-mains, respectively. Clustal W software was used foraligning genes and pertinent encoding sequences [28].
Recombinant protein expression and purificationThe pEXP5-PvARP recombinant plasmid which encodesthe entire PvARP sequence without the signal peptide (con-firmed by sequencing) was transformed in E. coli BL21-AI(Invitrogen), according to the manufacturer’s recommenda-tions. A protocol described by Sivashanmugam and hisgroup [29] with some modifications, was used for
improving expression yield. Briefly, the cells were grownovernight at 37°C in 10 mL Luria Bertani (LB) mediumcontaining 100 μg/mL ampicillin and 0.1% (w/v) D-glucose. The initial inoculum was then seeded in 100 mLLB volume with the same amount of the aforementionedampicillin and D-glucose and left to grow at 37°Cusing ~300 rpm until reaching 0.5 OD600; 0.2% L-arabin-ose (w/v) was used for five hours to induce expression.The culture was spun at 13,000 rpm for 30 min and lysedin extraction buffer (EB) (6 M urea, 12 mM imidazole,10 mM Tris-Cl, 100 mM NaH2PO4 and 10 mg/mL lyso-zyme) supplemented with protease inhibitors (1 mMPMSF, 1 mM iodoacetamide, 1 mM EDTA and 1 mg/mLleupeptin). PvARP recombinant expression (rPvARP) wasverified by Western blot and the protein was then purifiedby solid-phase affinity chromatography using Ni+2-NTAresin (Qiagen, California, USA) following the manufac-turer’s recommendations. Briefly, total lysate was incu-bated with the resin pre-equilibrated with EB overnight at4°C. The rPvARP mixture coupled to the resin was placedon a column and then washed several times with EB toeliminate weakly bound proteins. The recombinant pro-tein was eluted with EB containing imidazole at differingconcentrations (20, 100, 250 and 500 mM) in 3 mL frac-tions, which were analyzed by Coomassie blue staining toverify the presence of a single band and then dialyzedin PBS, pH 7.0. A micro BCA protein assay kit (ThermoScientific) was used for quantifying every fraction soobtained; a bovine serum albumin (BSA) curve was usedas reference.
Peptide synthesis and obtaining polyclonal antibodiesA 20 aa-long peptide (predicted to be a good B-cellepitope), located at the N-terminus of PvARP (CG-LDNLKAKESPSSNDDGVYAKG-GC), was synthesizedaccording to a previously-established methodology [30], po-lymerized, lyophilized and characterized by RP-HPLC andMALDI-TOF MS. Five mg of peptide (called 38582 herein)were immobilized on a CNBr-activated Sepharose 4B col-umn, according to the manufacturer’s recommendations. Apool of fifteen sera taken from patients who had sufferedprevious P. vivax malarial episodes (stored in FIDIC’sserum-bank, see the ‘Sample source’ section) was incubatedwith the peptide coupled to a Sepharose 4B column over-night at 4°C with constant shaking to purify specific anti-bodies against peptide 38582 (anti-PvARP38582). Theretained antibodies were eluted with gradients of increasingsalt concentration (50 mM-0.3 M NaCl); they were then di-alyzed in PBS, pH 7.8, and stored at −20°C until use.
SDS-PAGE and Western blotFive μg rPvARP and 50 μg total parasite proteins wereseparated on 12% SDS-PAGE and then transferred tonitrocellulose membranes. After having been blocked with
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5% skimmed milk in PBS-0.05% Tween for one hour, eachmembrane was cut into strips and individually analyzed asfollows: strips with the recombinant protein were incu-bated for two hours at room temperature (RT) with anti-PvARP38582 serum fractions (1:100 dilution) in a solutionof 5% skimmed milk in PBS-0.05% Tween to assess whichof them contained anti-PvARP specific antibodies; onestrip was incubated with an anti-histidine monoclonalantibody coupled to peroxidase (1:4,500) as positive con-trol for Western blot. Serum fractions recognizing the re-combinant protein were then used to detect PvARP intotal parasite lysate in the aforementioned conditions.Once antibody reactivity had been eliminated by incubat-ing anti-PvARP38582 serum with peptide 38582 for onehour at 37°C, then this solution was used as control. Fol-lowing three washes with PBS-0.05% Tween (5 min perwash), the strips were incubated for one hour withphosphatase-conjugated goat anti-human IgG as second-ary antibody (1:5,000) at RT. The blots were revealed witha VIP peroxidase (Vector Laboratories, Burlingame,Canada) or BCIP/NBT colour development substrate kits(Promega), according to the manufacturers’ indications.
Indirect immunofluorescence assay (IFA)Plasmodium vivax-parasitized reticulocytes were washedthrice with PBS and then diluted in this solution untilobtaining five to seven schizonts per field evaluated bystaining with Acridine orange. Twenty μL of the samplewere fed per well on eight-well multitest glass slides(Biomedicals, Inc) and the supernatant was removed10 min later. Once the samples were dry, they were fixedwith 4% formaldehyde for 5 min at RT. Following fivewashes with PBS, the sample was incubated with 1% Tri-ton X-100 for 5 min in the previously described condi-tions. After 10-min blocking at RT with 1% (v/v) skimmedmilk in PBS, each sample was incubated for one hour atRT with anti-PvARP38582 antibodies (20 μL). The sampleswere then incubated with FITC-conjugated anti-humanIgG antibody (Sigma) at 1:30 dilution for 45 min in thedark. The DNA was stained with DAPI (0.5 μg/mL) for10 min at RT and the excess was removed by washing sev-eral times with PBS-0.05% Tween. Once the slides hadbeen examined under an Olympus BX51 florescencemicroscope (using 100× oil immersion objective), Volocitysoftware (version 5.3.2) was used for superimposingthe images.
Enzyme-linked immunosorbent assay (ELISA)PvARP antigenicity was evaluated in triplicate usingserum from patients who had been living in malaria-endemic areas in Colombia and had presented episodesof such infection. Sera taken from healthy individuals whohad never suffered the disease were used as negative con-trols. Briefly, 96-well polysorb plates were covered with
1 μg/mL rPvARP overnight at 4°C and then incubatedat 37°C for one hour. The dishes were blocked with 200 μL5% skimmed milk - PBS-0.05% Tween for one hour at37°C. Antibody reactivity against the recombinant proteinwas evaluated by incubating the plates with a 1:100 dilutionof each human serum in 5% skimmed milk -PBS-0.05%Tween for one hour at 37°C. Following incubation of thedishes with peroxidase-coupled anti-human IgG secondaryantibody (1:10,000) diluted in 5% skimmed milk - PBS-0.05% Tween for one hour at 37°C, a peroxidase substratesolution (KPL Laboratories, WA, USA) was added to re-veal the reaction, according to the manufacturer’s recom-mendations. Optical density (OD) was detected at 620 nmwith an MJ ELISA multiskan reader and then calculated bysubtracting the OD value obtained from the control well(no antigen). A 0.11 cut-off value for evaluating the positiv-ity threshold was determined by taking the average of theOD plus twice the standard deviation (2 ± SD) of healthyindividuals’ sera reactivity.
Statistical analysisDifferences in average OD for rPvARP recognition byP. vivax-infected patients’ sera and in the control groupwere evaluated using the Kruskal-Wallis rank-sum test. A0.05 significance level was used for testing a statedhypothesis.
Sample sourceSera were obtained from 38 patients who were living inmalaria-endemic areas of Colombia and who had sufferedprevious episodes of P. vivax malaria (but not P. falcip-arum), as well as from 15 healthy individuals who hadnever been affected by the disease. All individuals signedan informed consent form after receiving detailed infor-mation regarding the study’s goals.
Accession numberThe nucleotide and aa sequences used here have beenreported in the GenBank database, under accessionnumber KC514070.
Results and discussionAnalyzing the arp gene in Plasmodium speciesThe P. vivax proteins identified as playing a potential rolein invasion by profile hidden Markov models [17] led toPvARP being selected. According to the information pro-vided by the PlasmoDB database, the Pvarp gene (accessnumber: PVX_090210) was found to be located betweenbase pairs 1,230,371 and 1,231,228 in chromosome 5 ofthe Sal-1 strain. Similar genes were also found in the gen-ome of other Plasmodium species known to be causingmalaria in humans (P. falciparum and Plasmodiumknowlesi), apes (Plasmodium cynomolgi) and rodents(Plasmodium berghei, Plasmodium yoelii and Plasmodium
Figure 1 Pvarp gene transcription during blood stage. Lane 1indicates the molecular weight marker. Lanes 2–4 show Pvarp geneamplification using cDNA RT+, RT- and gDNA, respectively. Lanes6–8 show the amplification of the Pvron1-a region using cDNA RT+,RT- and gDNA as template, respectively. Lane 5 shows the negativecontrol for amplifying the Pvarp gene.
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chabaudi). When analyzing alignment, the Pvarp gene co-dified product was 61.19%, identical to its orthologue inP. knowlesi (PKH_052690), 53.15% to its orthologuein P. cynomolgi (PCYB_053680) and 33.68% to itsorthologue in P. falciparum (PF3D7_0423400), while iden-tity ranged from 23.61% to 22.22% regarding orthologuesin P. chabaudi (PCHAS_052400), P. yoelii (PY06454) andP. berghei (PBANKA_052380). Such genes were located ina syntenic region, as corroborated by their open readingframe orientation and exon-intron structure. The forego-ing supported the idea that the Pvarp gene has been de-rived from a common ancestor; however, experimentalevidence concerning the functional role that the encodedprotein might have in different parasite species remains tobe determined.
The Pvarp gene is transcribed in schizontsThe presence of Pvarp gene transcripts in the P. vivaxVCG-1 strain was confirmed by PCR using the cDNA froma parasite sample as template. Figure 1 shows the Pvarpgene amplification products (excluding the signal peptide-encoding region) (lanes 2–4) and the Pvron1 gene’s a
Table 1 Mutations found in VCG-1 strain PvARP nucleotide an(Sal-1)
Base pairs* Amino acids* Mutations/Deletions
Changes in Pvarp nuin P. viva
Sal-I
456 152 Synonymous CAT
600-611 200-204 Deletion CATGAACGGAAA
650 216 Synonymous TAT
651 217 Non-synonymous GAA
655-656 218 Synonymous CGG
657 219 Non-synonymous AAA
663 221 Synonymous AAC
*Nucleotide and amino acid positions are numbered according to PvARP in the Sal-
region (lanes 5–8) from cDNA and gDNA. A ~810 bp band(Figure 1; lane 2) obtained from cDNA amplification (RT+)showed that the Pvarp gene was transcribed in theschizont-enriched sample, similar to that reported in thetranscriptional profile for the Sal-1 strain showing a max-imum transcription level after 35 hours of intra-erythrocytelife cycle [31]. It was also confirmed that the Pvarp genewas encoded by a single exon once the sequences obtainedfrom cDNA and gDNA products (Figure 1; lanes 2 and 4)had been aligned. The presence of a single ~1,053 bp bandin Pvron1-a PCR (Figure 1; lane 6) indicated that the cDNAhad not been contaminated by gDNA given that theexpected product for the latter would have been ~1,559 bp(Figure 1; lane 8). No amplification was observed in thenegative controls for each PCR (Figure 1; lanes 3 and 7(RT-), and 5 (DNA-free water)).Comparing Aotus monkey-adapted VCG-1 strain Pvarp
gene sequences to those from the Sal-1 reference strain ledto identifying four synonymous mutations, two non-synonymous ones producing aa changes (i e, methionine(M) for asparagine (N) and glycine (G) for N in aa position217 and 219, respectively) and a 12-base pair deletionrelated to an asparagine-methionine-asparagine-glycine(NMNG) repeat block (Table 1). It has been found thatparasite proteins have both highly polymorphic and con-served regions; the former are the target for an immune re-sponse while conserved sequences implicated in interactionwith cell receptors are usually not antigenic [32]. Consider-ing that the latter regions might be suitable targets forblocking parasite entry to host cells, further studies aimedat evaluating Pvarp gene polymorphism in different isolatesare required to determine which sequences could be usedas components of a vaccine against malaria caused byP. vivax.
Characterizing PvARP in silicoThe VCG-1 strain Pvarp gene encoded a 281 aa long pro-tein having ~30 kDa molecular mass, this being 64 residues
d amino acid sequences regarding the reference strain
cleotide sequencesx strains
Changes in the PvARP amino acid sequencesin P. vivax strains
VCG-1 Sal-I VCG-1
CAC H -
- NMNG -
TAA N -
CAA M N
CAA N -
CAA G N
AAT N -
I reference strain.
Figure 2 In silico characterization of PvARP, showing signal peptide localization, tandem repeats (TR), asparagine (ARR) and proline(PRR) amino acid repeat regions and the peptide selected for the antibody purification assay (shown in the box).
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longer when compared to its homologue PfAARP (217 aa)[18]. PvARP consists of 20% asparagine residues and has asignal peptide with a cleavage site between aa TNG-KS(Figure 2). A post-translational modification false positiveconsisting of a C-terminal glycosylphosphatidylinositol(GPI) anchor sequence has been predicted [17], differing toits P. falciparum homologue which has a true positive one.Asparagine- and proline-rich regions were found towardsthe C-terminal extreme of the protein sequence; the first ofthese covered residues 212 to 235, while the another onewas found downstream between aa 242 and 259 (Figure 2).Additionally, a tandem repeat region (TR), a feature sharedwith other vaccine candidates described to date, was alsofound using XSTREAM software [26] (Figure 2); this regionconsisted of 11 repeat blocks from the (D/N/S)(V/M)NGconsensus sequence found in aa 168 to 211. The sequencewas seen to be exclusive for P. vivax and had mutations(two substitutions and four deletions), thereby suggestingthat it was under pressure from the immune system. TRhave been common in several P. vivax antigens describedto date, which are mainly located on the surface or in apicalorganelles; these would include the circumsporozoite pro-tein (CSP) [33], merozoite surface protein 9 (MSP-9) [34],Pv34 [35] and rhoptry neck proteins 1 and 2 [23,36]. Even
Figure 3 Detecting recombinant and parasite protein by human antibshow non-induced and induced cell lysate, respectively (Coomassie staininanalyzed by Western blot using anti-polyhistidine antibodies, respectively. (PvARP by Western blot, respectively. Lane 2 shows the absence of humanindicates PvARP recognition. Lane 4 shows detection of recombinant protein kDa.
though several studies have shown that the tandem repeatsof PvCSP trigger an immune response when inoculated inprimates and humans [33,37,38], the response so produceddid not completely inhibit infection caused by the parasite.It has been shown in other Plasmodium species that TRcould act as a smokescreen against the immune system,thereby diverting strong reactions towards functionally-relevant regions [39]; however, their exact role in P. vivaxantigens remains unknown.
PvARP expression in schizonts and subcellular localizationSpecific human antibodies against an N-terminal PvARPsynthetic peptide (Figure 2) were used for checking pro-tein expression and localization in the schizont-enrichedsample. PvARP was recombinantly expressed excludingthe signal peptide and then purified (Figure 3A). Once hu-man anti-PvARP38582 antibody ability to detect the recom-binant protein in Western blot assays had been checked(Figure 3B), they were then used for detecting the proteinon a blot containing parasite total lysate (Figure 3C). Boththe parasite and recombinant PvARP proteins weredetected above the expected weight (~40 and ~49 kDa, re-spectively), probably due to the presence of acidic aa(aspartic acid and glutamic acid) thereby causing
odies. (A) Recombinant protein expression and purification. Lanes 2–3g). Lanes 4–5 show purified rPvARP stained with Coomassie orB and C) Antibody ability to recognize recombinant and parasiteserum reactivity after being pre-incubated with peptide 38582. Lane 3in (positive control). MW kDa indicates molecular weight marker
Figure 4 PvARP sub-cellular localization in mature schizonts. (A) Shows the detection of the protein on free merozoite surface. (B) PvARPlabelling on mature schizonts. The nuclei are labelled with DAPI (blue). An amplified image of a merozoite (indicated by an arrow) is shown insmall boxes.
Figure 5 rPvARP antigenicity. The box diagram shows ODdistribution (Y axis) for detecting rPvARP by sera from non-infected andinfected individuals (X axis). *: Infected individuals (n = 38; X̄±S = 0.5 ±0.2; 95%CI = 0.16-1.1) and control (n = 15; X̄±S = 0.1 ± 0.07; 95%IC =0.03-0.24). p value = 0.000.
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anomalous migration on SDS-PAGE gel. The antibodieshad specific reactivity to a ~40 kDa band; such reactivitywas eliminated by using serum which had been pre-incubated with peptide 38582 (Figure 3C; lane 2).A strong fluorescence signal, having an apparent con-
centration towards the apical pole, was found on free mer-ozoites’ surface and in mature schizonts when using theserum as primary antibody in the parasitized reticulocytesample (Figure 4). The results led to the suggestion thatPvARP could be expressed in apical organelles and thenbecome relocated to the surface. However, other confocalor electron microscope assays are needed to determinethe protein’s exact localization pattern.
Antigenicity in humansPvARP antigenic ability was evaluated by ELISA, using thesera from 38 patients who had suffered P. vivax malariaand 15 serum samples from people who had never sufferedfrom the disease. The statistical test revealed a statisticallysignificant difference between the medians (m) of thegroups (Wilcoxon rank-sum test. Z = 5.1, p = 0.000); it gavem = 0.5 for the group of infected patients and m = 0.1 forthe control group (Figure 5), thereby corroborating the factthat the protein was able to trigger an antibody response inthe host during natural P. vivaxmalaria infection, most serabeing able to recognize native and recombinant protein, asdemonstrated by IFA and Western blot, respectively. Theresults supported the idea of analyzing this protein’s poten-tial as a candidate for an anti-P. vivax vaccine.
ConclusionsThis study has described how the P. vivax asparagine-rich protein was characterized. As demonstrated, PvARP
was conserved among different species belonging to thePlasmodium genus and shared some features of well-characterized surface and/or apical proteins being studiedas candidates for a vaccine, such as prominent transcrip-tion and expression towards the end of the intra-erythrocyte life cycle and broad recognition by sera frompatients infected with P. vivax malaria. The results sup-ported the notion that this antigen could be a promisingcandidate for inclusion when developing an anti-malarialvaccine. Further immunogenicity assays and studies of theability to induce protection in the experimental Aotusmodel are required.
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Competing interestsThe authors declare that they have no competing interests.
Authors’ contributionsDAMP designed experiments, analyzed data and wrote the initial manuscript.AS carried out molecular biology and immunochemical assays. MAPdesigned, evaluated and coordinated the assays and corrected the finalmanuscript. All authors read and approved the final manuscript.
AcknowledgementsWe would like to thank Daniela Prieto Borja for her technical support, JasonGarry for translating and reviewing this manuscript and especially ProfManuel Elkin Patarroyo for his invaluable comments and suggestions. Thisresearch was supported by the “Instituto Colombiano para el Desarrollo de laCiencia ‘Francisco José de Caldas” (COLCIENCIAS) contract RC#309-2013.
Received: 28 January 2013 Accepted: 12 May 2013Published: 20 May 2013
References1. WHO: World malaria report. Geneva: World Health Organization; 2011.2. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F,
Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K,Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NP, Lindegardh N,Socheat D, White NJ: Artemisinin resistance in Plasmodium falciparummalaria. N Engl J Med 2009, 361:455–467.
3. Hunt RH, Fuseini G, Knowles S, Stiles-Ocran J, Verster R, Kaiser ML, Choi KS,Koekemoer LL, Coetzee M: Insecticide resistance in malaria vectormosquitoes at four localities in Ghana, West Africa. Parasit Vectors 2011, 4:107.
4. Guerra CA, Howes RE, Patil AP, Gething PW, Van Boeckel TP, Temperley WH,Kabaria CW, Tatem AJ, Manh BH, Elyazar IR, Baird JK, Snow RW, Hay SI: Theinternational limits and population at risk of Plasmodium vivaxtransmission in 2009. PLoS Negl Trop Dis 2010, 4:e774.
5. Genton B, D’Acremont V, Rare L, Baea K, Reeder JC, Alpers MP, Muller I:Plasmodium vivax and mixed infections are associated with severemalaria in children: a prospective cohort study from Papua New Guinea.PLoS Med 2008, 5:e127.
6. Tjitra E, Anstey NM, Sugiarto P, Warikar N, Kenangalem E, Karyana M,Lampah DA, Price RN: Multidrug-resistant Plasmodium vivax associatedwith severe and fatal malaria: a prospective study in Papua, Indonesia.PLoS Med 2008, 5:e128.
7. Barrero CA, Delgado G, Sierra AY, Silva Y, Parra-Lopez C, Patarroyo MA:Gamma interferon levels and antibody production induced by twoPvMSP-1 recombinant polypeptides are associated with protectiveimmunity against P. vivax in Aotus monkeys. Vaccine 2005, 23:4048–4053.
8. Sierra AY, Barrero CA, Rodriguez R, Silva Y, Moncada C, Vanegas M,Patarroyo MA: Splenectomised and spleen intact Aotus monkeys’immune response to Plasmodium vivax MSP-1 protein fragments andtheir high activity binding peptides. Vaccine 2003, 21:4133–4144.
9. Xue X, Ding F, Zhang Q, Pan X, Qu L, Pan W: Stability and potency of thePlasmodium falciparum MSP1-19/AMA-1(III) chimeric vaccine candidatewith Montanide ISA720 adjuvant. Vaccine 2010, 28:3152–3158.
10. Gentil F, Bargieri DY, Leite JA, Francoso KS, Patricio MB, Espindola NM, VazAJ, Palatnik-de-Sousa CB, Rodrigues MM, Costa FT, Soares IS: A recombinantvaccine based on domain II of Plasmodium vivax Apical MembraneAntigen 1 induces high antibody titres in mice. Vaccine 2010,28:6183–6190.
11. Dicko A, Diemert DJ, Sagara I, Sogoba M, Niambele MB, Assadou MH,Guindo O, Kamate B, Baby M, Sissoko M, Malkin EM, Fay MP, Thera MA,Miura K, Dolo A, Diallo DA, Mullen GE, Long CA, Saul A, Doumbo O, MillerLH: Impact of a Plasmodium falciparum AMA1 vaccine on antibodyresponses in adult Malians. PLoS One 2007, 2:e1045.
12. Herrera S, Bonelo A, Perlaza BL, Valencia AZ, Cifuentes C, Hurtado S,Quintero G, Lopez JA, Corradin G, Arevalo-Herrera M: Use of long syntheticpeptides to study the antigenicity and immunogenicity of the Plasmodiumvivax circumsporozoite protein. Int J Parasitol 2004, 34:1535–1546.
17. Restrepo-Montoya D, Becerra D, Carvajal-Patino JG, Mongui A, Nino LF,Patarroyo ME, Patarroyo MA: Identification of Plasmodium vivax proteinswith potential role in invasion using sequence redundancy reductionand profile hidden Markov models. PLoS One 2011, 6:e25189.
18. Wickramarachchi T, Devi YS, Mohmmed A, Chauhan VS: Identification andcharacterization of a novel Plasmodium falciparummerozoite apical proteininvolved in erythrocyte binding and invasion. PLoS One 2008, 3:e1732.
protein sequence analysis software with client/server capabilities.Comput Biol Med 2001, 31:259–267.
21. Pico de Coana Y, Rodriguez J, Guerrero E, Barrero C, Rodriguez R, MendozaM, Patarroyo MA: A highly infective Plasmodium vivax strain adapted toAotus monkeys: quantitative haematological and moleculardeterminations useful for P. vivax malaria vaccine development.Vaccine 2003, 21:3930–3937.
22. Andrysiak PM, Collins WE, Campbell GH: Concentration of Plasmodiumovale- and Plasmodium vivax-infected erythrocytes from nonhumanprimate blood using Percoll gradients. Am J Trop Med Hyg 1986,35:251–254.
23. Moreno-Perez DA, Montenegro M, Patarroyo ME, Patarroyo MA:Identification, characterization and antigenicity of the Plasmodium vivaxrhoptry neck protein 1 (PvRON1). Malar J 2011, 10:314.
25. Poisson G, Chauve C, Chen X, Bergeron A: FragAnchor: a large-scalepredictor of glycosylphosphatidylinositol anchors in eukaryote proteinsequences by qualitative scoring. Genomics Proteomics Bioinformatics 2007,5:121–130.
26. Newman AM, Cooper JB: XSTREAM: a practical algorithm for identificationand architecture modeling of tandem repeats in protein sequences.BMC Bioinformatics 2007, 8:382.
27. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P,Das U, Daugherty L, Duquenne L, Finn RD, Gough J, Haft D, Hulo N, Kahn D,Kelly E, Laugraud A, Letunic I, Lonsdale D, Lopez R, Madera M, Maslen J,McAnulla C, McDowall J, Mistry J, Mitchell A, Mulder N, Natale D, Orengo C,Quinn AF, Selengut JD, Sigrist CJ, Thimma M, Thomas PD, Valentin F, WilsonD, Wu CH, Yeats C: InterPro: the integrative protein signature database.Nucleic Acids Res 2009, 37:D211–D215.
28. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving thesensitivity of progressive multiple sequence alignment throughsequence weighting, position-specific gap penalties and weight matrixchoice. Nucleic Acids Res 1994, 22:4673–4680.
29. Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q: Practicalprotocols for production of very high yields of recombinant proteinsusing Escherichia coli. Protein Sci 2009, 18:936–948.
30. Houghten RA: General method for the rapid solid-phase synthesis oflarge numbers of peptides: specificity of antigen-antibody interaction atthe level of individual amino acids. Proc Natl Acad Sci U S A 1985,82:5131–5135.
31. Bozdech Z, Mok S, Hu G, Imwong M, Jaidee A, Russell B, Ginsburg H, NostenF, Day NP, White NJ, Carlton JM, Preiser PR: The transcriptome ofPlasmodium vivax reveals divergence and diversity of transcriptionalregulation in malaria parasites. Proc Natl Acad Sci U S A 2008,105:16290–16295.
32. Rodriguez LE, Curtidor H, Urquiza M, Cifuentes G, Reyes C, Patarroyo ME:Intimate molecular interactions of P. falciparum merozoite proteinsinvolved in invasion of red blood cells and their implications for vaccinedesign. Chem Rev 2008, 108:3656–3705.
33. Yang C, Collins WE, Xiao L, Saekhou AM, Reed RC, Nelson CO, Hunter RL,Jue DL, Fang S, Wohlhueter RM, Udhayakumar V, Lal AA: Induction ofprotective antibodies in Saimiri monkeys by immunization with amultiple antigen construct (MAC) containing the Plasmodium vivaxcircumsporozoite protein repeat region and a universal T helper epitopeof tetanus toxin. Vaccine 1997, 15:377–386.
Moreno-Pérez et al. Malaria Journal 2013, 12:165 Page 9 of 9http://www.malariajournal.com/content/12/1/165
34. Oliveira-Ferreira J, Vargas-Serrato E, Barnwell JW, Moreno A, Galinski MR:Immunogenicity of Plasmodium vivax merozoite surface protein-9recombinant proteins expressed in E. coli. Vaccine 2004, 22:2023–2030.
35. Mongui A, Angel DI, Gallego G, Reyes C, Martinez P, Guhl F, Patarroyo MA:Characterization and antigenicity of the promising vaccine candidatePlasmodium vivax 34 kDa rhoptry antigen (Pv34). Vaccine 2009, 28:415–421.
37. Herrera S, De Plata C, Gonzalez M, Perlaza BL, Bettens F, Corradin G, Arevalo-Herrera M: Antigenicity and immunogenicity of multiple antigenpeptides (MAP) containing P. vivax CS epitopes in Aotus monkeys.Parasite Immunol 1997, 19:161–170.
38. Herrera S, Bonelo A, Perlaza BL, Fernandez OL, Victoria L, Lenis AM, Soto L,Hurtado H, Acuna LM, Velez JD, Palacios R, Chen-Mok M, Corradin G,Arevalo-Herrera M: Safety and elicitation of humoral and cellularresponses in colombian malaria-naive volunteers by a Plasmodium vivaxcircumsporozoite protein-derived synthetic vaccine. Am J Trop Med Hyg2005, 73:3–9.
